This invention relates to the immunotherapy of disease states, and in particular, but not exclusively to the immunotherapy of carcinomas.
Cancer is a major cause of death and severe trauma in modern society. Cancer is no respecter of persons as the young, old, males, females and peoples of all races may contract cancer, although cancer in children is relatively rare, perhaps with the exception of childhood leukemia. In western society, cancer of the colon and lung cancer are major diseases. In women, breast cancer is the most common form of cancer.
Many cancers are accompanied by overproduction of human mucin. Mucins are heavily glycosylated proteins (greater than about 100 Kd) which are produced by many epithelial cells and tumours (1). Mucins found on cancer cells are different in some respects to those on normal epithelial cells, in that some mucins have a deficiency in their carbohydrate coat which leaves the protein core exposed. (2). There are seven forms of known human mucin designated MUC1, MUC2, MUC3, MUC4, MUC5 MUC6 and MUC7 (3, 4, 26, 27). MUC1 is the most ubiquitous. The various mucins all have very similar properties, that is, they are transmembrane glycoproteins, all having a variable number of repeated amino acid sequences, which have a high content of serine, threonine and proline. Overproduction of aberrantly glycosylated mucins (either non-glycosylated or a deficiency in glycosylation) is characteristic of tumours of the breast, ovary, pancreas, colon, lungs, prostate and other tumours of secretory tissue. The cDNA sequences of the respective protein cores of the human mucins MUC1 to MUC7 have been cloned and characterized and have been found to contain highly repetitive central portions of varying numbers of repeats of particularly amino acid motifs (known as VNTR""s). By way of example, MUC1 consists of unique amino and carboxyl terminal sequences separated by a highly repetitive central portion containing forty to eighty tandemly arranged copies or repeats of a twenty amino acid motif. The VNTR""s of MUC1 through MUC7 are set forth below:
The repeated subunit of MUC6 comprises 169 amino acids, although at this time the amino acid sequence of this repeat unit has not been fully characterized. The MUC7 sequence has recently been published (27).
Finn and colleagues have demonstrated that in the lymph nodes of patients with breast cancer (5, 6), cancer of the pancreas, ovary and other tumours, cytotoxic lymphocytes are present which react with human mucin. Antibodies to the MUC1 peptide can block the activity of these cytotoxic T-lymphocytes on MUC1+ target cells (5, 6). Recently, cytotoxic lymphocytes to a murine lung cancer have also been described (28).
The surgery associated with tumour removal is traumatic to the patient, often disfiguring, and costly. Established chemotherapeutic and radiation procedures for tumour treatment which may be carried out in place of or in conjunction with surgical procedures are often debilitating and associated with severe side-effects. There is accordingly an urgent need for therapeutic compounds and methods for the prevention/treatment of tumours.
There is an urgent need for new compounds and methods for the treatment of cancer. Similarly, there is a pressing need for alternative compounds and therapies for the treatment of other disease states such as type I allergies, malaria, HLV, dental caries, flu, cholera, foot and mouth disease, meningitis, Leishmania infection, whooping cough, rabies, Streptococcus infection, respiratory infection, measles, Lyme disease, tuberculosis, bacterial meningitis, shingles, rubella, hepatitis, herpes, hepatitis A, polio, venereal disease/trachoma, hepatitis B, common cold, cervical cancer, meningitis/pneumonitis, chicken pox, small pox, pneumonia/PUO.
In accordance with the first aspect of the present invention, there is provided a compound comprising a conjugate between an antigen and a carbohydrate polymer.
In accordance with another aspect of the present invention, there is provided a compound comprising a conjugate between the human mucin polypeptide, one or more repeated subunits thereof, or a fragment of said repeated subunits, with a carbohydrate polymer.
In a preferred embodiment of the present invention, the carbohydrate polymer is a polymer of the carbohydrate mannose.
Insofar as the present invention is concerned, the antigen can be a human autoantigen or a peptide antigen derived from a virus, microorganism or plant or an amino acid subunit of at least five amino acids in length of a human autoantigen or a peptide antigen derived from a virus, microorganism or plant. The antigen of the present invention can also consist of more than one, five or more amino acid subunits (as mentioned above) linked together. These linked subunits may be from the same or different origins within the bounds described above.
Examples of the antigens envisaged by the present invention are as follows: pollens, hepatitis C virus (HIV) core, E1, E2 and NS2 proteins, Plasmodium faliciparum circumsporozoite protein, HIV-gp1201160 envelope glycoprotein, streptococcus surface protein Ag, influenza nucleoprotein, haemagglutinin-neuraminidase surface infection, TcpA pilin subunit, VP1 protein. LMCV nucleoprotein, Leishmania major surface glycoprotein (gp63), Bordetella pertussis surface protein, rabies virus G protein, Streptococcus M protein, Syncyticial virus (RSV) F or G proteins, Epstein Barr virus (EBV) gp340 or nucleoantigen 3A, haemagglutinin, Borrelia burgdorferi outer surface protein (Osp) A, Mycobacterium tuberculosis 38 kDa lipoprotein or Ag85, Neisseria meningitidis class 1 outer protein, Varicella zoster virus IE62 and gp1, Rubella virus capsid protein, Hepatitis B virus pre S1 ag, Herpes simplex virus type I glycoprotein G or gp D or CP27, Murray valley encephalitis virus E glycoprotein, Hepatitis A virus VP1, polio virus capsid protein VP1, VP2 and VP3, chlamydia trachomatis surface protein, Hepatitis B virus envelope Ag pre S2, Human rhinovirus (HRV) capsid, papillomavirus peptides from oncogene E6 and E7, Listeria surface protein, Varicella virus envelope protein, Vaccinia virus envelope protein, Brucella surface protein, a combination of one or more of said antigens, an amino acid subunit of said antigens comprising five or more amino acids in length or combinations of one or more of said subunits.
The antigens of the present invention can also consist of whole cells or sub-cellular fractions thereof. Such cells or sub-cellular fractions thereof may be derived from any tumour type or other source. Examples of cancer types from which the whole cells or sub-cellular fractions may be derived are breast, lung, pancreas and colon cancer and melanoma. Some further examples of specific antigens obtained from tumours are melanoma specific antigen (for example, the MAGE series antigen), carcino embryonic antigen (CEA) from colon and other cancers or indeed antigens extracted from any tumour.
This invention includes any one or more of the antigens listed and in particular includes any one ore more of the human mucins MUC1 through MUC7 which, as mentioned above, all comprise highly repetitive central portions of repeated amino acid sequences which are high in serine, threonine and proline. In particular, the compounds of this invention may comprise a human mucin polypeptide (containing a variable number of repeats associated with normal allelic variation), or may comprise one or more of the repeated sequences of human mucin, preferably two to eighty, more preferably two to twenty and even more preferably two to ten repeated subunits of human mucin or it may comprise the whole native MUC1 molecule. The human mucin and subunits thereof are preferably non-glycosylated or aberrantly glycosylated so as to provoke an immune response to the mucins found on cancer cells which have a deficiency in their carbohydrate coat which leaves the protein core exposed. The use of human mucin MUC1 is particularly preferred although it is to be clearly understood that the invention extends to the use of any antigen and especially to the use of the human mucins MUC1 through MUC7. For the purpose of convenience, the term MUC will hereafter be used to refer to any of the human mucins MUC1 through MUC6 and repeated subunits thereof. While only the human mucins will be dealt with hereafter, it must be kept in mind that this invention equally relates to any other antigen as mentioned previously.
Fragments of MUC may also be conjugated to a carbohydrate polymer. These fragments would generally comprise from five to twenty amino acids from the repeated amino acid sequences of any mucins MUC1 through MUC6. For example, a fragment of the mucin MUC1 may comprise the amino acid sequence APDTR SEQ ID NO: 7, APDTRPAPG SEQ ID NO: 8, DTRPAPGSTAPP SEQ ID NO: 9, and the like. For convenience of description these fragments are also included with the definition MUC. Similarly, other antigen fragments comprising at least five amino acids may be conjugated to a carbohydrate polymer.
A specified antigen (such as MUC1, MUC2, MUC3, MUC4, MUC5, MUC6 or MUC7) may form part of a fusion protein in order to facilitate expression and purification on production of the fusion protein in recombinant host cells. The non-antigen portion of the fusion protein would generally represent the N-terminal region of the fusion polypeptide with the carboxy terminal sequences comprising antigen sequences. Fusion proteins may be selected from glutathione-S-transferase, xcex2-galactosidase, or any other protein or part thereof, particularly those which enable affinity purification utilizing the binding or other affinity characteristics of the protein to purify the resultant fusion protein. The protein may also be fused to the C-terminal or N-terminal of the carrier protein. The nature of the fusion protein will depend upon the vector system in which fusion proteins are produced. An example of a bacterial expression vector is pGEX which on subcloning on a gene of interest into this vector produces a fusion protein consisting of glutathione-S-transferase with the protein of interest. Examples of other vector systems which give rise to fusion proteins with a protein of interest are described in Sambrook et al (7), which is incorporated herein in its entirety by reference. These can be included or cleaved; if included they could a have a xe2x80x9ccarrierxe2x80x9d function.
The protein or fusion protein maybe expressed in a number of prokaryotic (E. coli or xcex2-sutilis) or eukaryotic (baculovirus, CHO cells, cos cells or yeast) expression systems. In some of these systems, for example, baculovirus or yeast, by introducing glycosylation motifs into the protein or fusion protein, the mannose rich glycosylation may be adequate; negating the need for chemically linking with mannose rich carbohydrate polymers. These novel fusion proteins may be used with or without mild periodate oxidation.
The carbohydrate portion of the compounds of the invention may comprise any carbohydrate polymer, for example, selected from polymers of glucose, galactose, mannose, xylose, arabinose, fucose, glucosamine, galactosamine, rhamnose, 6-O-methyl-D-galactose, 2-O-acetyl-xcex2-D-xylose, N-acetyl-glucosamine, iduronate, guluronate, mannuronate, methyl galacturonate, xcex1-D-galactopyranose 6-sulphate, fructose and xcex1 abequose, conformation and configuration isomers thereof, or a carbohydrate formed of two or more different monomer units. The number of repeated monomer units in the polymer is not important but generally carbohydrate polymers would comprise at least twenty monomer units, preferably in excess of one hundred monomer units, more preferably in excess of one thousand monomer units, and still more preferably in excess of ten thousand monomer units or more. Carbohydrate polymers may be a mixture of polysaccharide chains of varying molecular weights. Most preferably the carbohydrate polymer is a polymer of mannose or is a carbohydrate polymer containing mannose units.
Antigens may be conjugated to a carbohydrate polymer according to standard processes well known in the art of carbohydrate chemistry for the derivatization and reaction of polysaccharides and monosaccharides. Carbohydrates may be oxidized with conventional oxidizing reagents such as sodium periodate to give a polyaldehyde which is then directly reacted with the antigen (such as repeated subunits of MUC1) where amino functional groups on the protein chain (such as the xcex5 group of lysine) react with the aldehyde groups which may optionally be further reduced to form a Schiff base. Polysaccharide chains may be first activated with cyanogen bromide and the activated polysaccharide then reacted with a diamine, followed by conjugation to the antigen to form a conjugate which may optionally then be oxidized. The carbohydrate and polypeptide may be derivatized with bifunctional agents in order to cross-link the carbohydrate and polypeptide. Commonly used cross-linking agents include 1,1-bis(diazoacetyl}-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicyclic acid, homobifunctional imidoesters including disuccinimidyl esters such as 3,3xe2x80x2-dithiobis(succinimidyl-propionate), and bifunctional maleimides such as bis-N-maleimido-1, 8-octane. Derivatizing agents such as methyl-3-[(p-azido-phenyl)dithio]propioimidate yield photactivitable intermediates which are capable of forming cross-links in the presence of light. Oxidized carbohydrates may be reacted with hydrazine derivatives of antigens to give a conjugate. Alternatively, carbohydrates may be reacted with reagents such as carbonyl diimidazole, which after oxidation gives the desired conjugate.
The coupling of antigens to a carbohydrate involves converting any or all of the functional groups on the carbohydrate to reactive groups and thereafter reacting the reactive groups on the carbohydrate with reactive groups on the polypeptide. Carbohydrate polymers are replete with hydroxide groups, and in some instances, carboxyl groups (such as in idruionate), ester groups (such as methylgalacturonate) and the like. These groups may be activated according to standard chemical procedures. For example, hydroxyl groups may be reacted with hydrogen halides, such as hydrogen iodide, hydrogen bromide and hydrogen chloride to give the reactive halogenated polysaccharide. Hydroxy groups may be activated with phosphorous trihalides, active metals (such as sodium ethoxide, aluminium isopropoxide and potassium tert-butoxide), or esterified (with groups such as tosyl chloride or acetic acid) to form reactive groups which can be then be reacted with reactive groups on the polypeptide to form one or more bonds. Other functional groups on carbohydrates apart from hydroxyl groups may be activated to give reactive groups according to well known procedures in the art.
Polypeptides comprising MUC or other antigens may be produced according to well known procedures such as peptide synthesis, protein purification, or expression of polypeptides in host cells. Peptide synthesis may be employed for polypeptides containing up to about a hundred amino acids (for example, five repeated subunits of MUC1). Generally, for polypeptide containing about twenty or more amino acids, the preferred means of production is recombinant expression in a host cell, preferably a prokaryotic host cell, and more preferably a bacterial host cell. However, as discussed earlier, eukaryotic systems may also be used. Procedures for expression of recombinant proteins in host cells are well established, see, for example, Sambrook, et al (7).
Carbohydrates may be purified from natural sources or synthesized according to conventional procedures. Carbohydrates are available commercially from many suppliers. For example, the antigens of the invention may be coupled to keyhole limpet hemacyanin (KLH) using glutaraldehyde and then reacted with oxidised mannan.
In another aspect, the invention relates to an immunogenic vaccine against human disease states and in particular against tumour cells expressing human mucin or a subunit thereof, which comprises a compound comprising a conjugate between an antigen and a carbohydrate polymer, in association with a pharmaceutically acceptable carrier. Antigens which may be used in this aspect of the invention are as previously described. The vaccine is administered to human patients to protect against various disease states including cancer cell growth, and in particular, the growth of tumours of secretory tissues, such as tumours of the breast, colon, lung, pancreas, prostate, and the like. Patients may be immunized with the vaccine to protect against tumour formation of secretory tissues. Alternatively, patients suffering from tumours may be immunized with the vaccine as part of a therapeutic regimen for tumour treatment. By way of example, to protect women from breast cancer, women may be immunized with the vaccine pre- or post-puberty and may receive one or more injections, preferably an initial immunization, followed by one or more booster injections separated by several months to several years. In one immunization schedule, women may be immunized with the compounds of the invention and then receive a booster immunization at appropriate intervals. Further booster immunizations are then provided at regular intervals. The route of immunization is no different from conventional human vaccine administration. Accordingly, vaccines may be administered subcutaneously, intramuscularly, orally, intravenously, and the like.
In a particularly preferred aspect the invention provides a immunogenic vaccine against tumour cells expressing human mucin which vaccine comprises a conjugate between whole human milk fat globule antigen (HMFG) and a carbohydrate polymer in association with a pharmaceutically acceptable carrier.
Some other disease states which may be protected against in this manner include, type I allergies, malaria, HIV, dental caries, flu, cholera, foot and mouth disease, meningitis, Leishmania infection, whooping cough, rabies, Streptococcus infection, respiratory infection, measles, Lyme disease, tuberculosis, bacterial meningitis, shingles, rubella, hepatitis, herpes, hepatitis A, polio, venereal disease/trachoma, hepatitis B, common cold, cervical cancer, meningitis/pneumonitis, chicken pox, small pox, pneumonia/PUO.
The amount of compounds of the invention or compositions thereof delivered to a patient is not critical or limiting. An effective amount of a compound of the invention is that which will stimulate an immune response against the antigen component. The amount of compounds or compositions delivered may vary according to the immune status of the patient (depending on whether the patient is immunosuppressed or immunostimulated), the judgement of attending physician or veterinarian whether the compound is used as a vaccine to prevent or treat a disease state or as a vaccine to prevent tumour formation, or whether the vaccine is used in the treatment of an existing tumour. By way of example, patients may receive from 1 xcexcg to 10,000 xcexcg of the compounds of the invention, more preferably 50 xcexcg to 5,000 xcexcg, still more preferably 100 xcexcg to 1,000 xcexcg, and even more preferably 100 xcexcg to 500 xcexcg of the compounds of the invention. Adjuvants are not generally required. However, adjuvants may be used for immunization. Suitable adjuvants include alum, as well as any other adjuvant or adjuvants well known in the vaccine art for administration to humans.
Compounds of the invention may be administered to patients in concert with a cytokine or other immune regulator. By way of example, immune regulators which may be administered in concert with the compounds of the invention include one or more of GM-CSF, G-CSF, M-CSF, TNFxcex1 or xcex2, interferon xcex1 or xcex3, any of IL1 through IL13, or any other cytokine. The immune regulator may be administered at the same time as the compounds of the invention, optionally as part of a multi-component administration form. Alternatively, the compounds of this invention and immune regulators may be administered at different time intervals.
In a still further aspect of this invention, there is provided a method for inducing a cell mediated immune response against antigens which comprises administering to an animal (including a human) a compound comprising a conjugate between said antigen and a carbohydrate polymer, optionally in association with a pharmaceutically acceptable carrier.
The immunization of humans and animals with the compounds of this invention may provoke a potentiated cellular response of activated T-lymphocytes which are cytotoxic to cells expressing the antigen component. By way of example, humans and animals may be immunized against tumours which express human mucins. A potential benefit of this invention arises from the fact that animals may be protected against cancer prior to tumour growth, as the compounds of the invention may provoke a cellular immune response of cytotoxic T-cells which kill tumour cells expressing mucin or other antigenic determinants. This invention is applicable to the immunization against tumours of secretory tissue, such as adenocarcinomas, more particularly, tumours of the breast, ovary, pancreas, colon, lung, prostate and the like.
The compounds of the invention may also be used as therapeutic agents for the treatment of patients suffering from cancer, as a part of the overall treatment for eradication of the cancer. Thus, the compounds of the invention may be administered to patients suffering from cancer either before or after surgery to remove the tumour. Preferably the compounds are administered as part of a chemotherapeutic regime following tumour excision. In these circumstances, the compounds of the invention are administered in amounts consonant with standard chemotherapeutic regimes for the administration of cytotoxic compounds for use in tumour treatment.
The compounds of this invention can also be used In immunization for therapy or prophylaxis of other disease states as mentioned earlier.
In a still further aspect, the invention relates to the use of a compound comprising a conjugate between the human mucin polypeptide, one or more repeated subunits thereof, or a fragment of said repeated subunits and a carbohydrate polymer in the treatment of adenocarcinoma, particularly breast cancer.
The compounds of this invention possess the advantage of being substantially non-toxic on administration to animals or humans, and as a consequence the compounds are well tolerated by administration to patients.
The invention described herein is not restricted to the human mucin MUC1. The invention clearly extends to the use of other mucins expressed by cancer cells, as well as to the use of other antigens which on coupling to polysaccharides, can be used to provoke cytotoxic T-cell responses against tumour cells, which compounds may be used in vaccines to prevent tumour formation, as well as for the treatment of cancer, and/or the treatment or prophylaxis of other disease states as mentioned earlier.
In a further aspect the invention provides an immunogenic peptide, protein or portion thereof capable of eliciting an immune response comprising an amino acid sequence of non VNTR, non leader regions of mucin.
The immunogenic peptide, protein or portion thereof may be derived from natural sources, synthesized according to standard techniques or produced recombinantly. The immunogenic peptide, protein or portion thereof may be part of a fusion protein.
The immunogenic peptide, protein or portion thereof may be produced according to well known procedures such as those described earlier.
Preferably the immunogenic peptide, protein or portion thereof is derived from human mucin 1, or the amino acid sequence is based on that of a human mucin 1. More preferably the immunogenic peptide, protein or portion thereof is derived from or the amino acid sequence is based on human milk fat globule membrane antigen (HMFG).
Even more preferably the immunogenic peptide, protein or portion thereof is derived from the extracellular region or intracellular region of human MUC1.
Even more preferably the immunogenic peptide, protein or portion thereof is glycosylated. It will be understood by a person skilled in the art that one or more amino acids of the immunogenic peptide, protein or portion thereof may be suitably glycosylated.
Still more preferably the immunogenic peptide, protein or portion thereof comprises one of the following amino acid sequence or an immunogenic fragment thereof:
The immunogenic peptide, protein or portion thereof may be a mutant variant or derivative of amino acid sequences (i) to (ix) above, provided it has the same or similar immunogenic properties.
The inventors"" data suggest that the non-VNTR regions of MUC1 may be advantageous immunologically as they appear to steer antigen presentation towards stimulation of the cellular immune response. By contrast, the VNTR region can deviate the immune response towards antibodies due to cross-reaction with the gal antibodies. The preferential stimulation of cellular immune responses e.g. upregulation of cytotoxic T cells etc could provide efficacious and long-lasting non-VNTR vaccination which would be highly advantageous.
In accordance with a further aspect of the present invention, there is provided a compound comprising a conjugate between the immunogenic peptide, protein or portion thereof described above and a carbohydrate polymer.
In a preferred embodiment of the present invention, the carbohydrate polymer is a polymer of the carbohydrate mannose.
The immunogenic peptides, proteins or portions thereof may have amino acid sequences which are derived from or based upon antigens from any tumour type or other source expressing MUC1. Examples of cancer types from which the whole cells or sub-cellular fractions may be derived from or based upon antigens from any tumour type or other source expressing MUC1. Examples of cancer types from which the whole cells or sub-cellular fractions may be derived are breast, lung, pancreas and colon cancer. Some further examples of specific antigens obtained from tumours are carcinoembryonic antigen (CEA) from colon and other cancers or indeed antigens extracted from any tumour expressing MUC1.
Fusion proteins may be selected from those described earlier.
The carbohydrate portion of the compounds of the invention may comprise those described earlier.
Antigens in the form of the immunogenic peptide, protein or portion thereof may be conjugated to a carbohydrate polymer according to standard processes well known in the art of carbohydrate chemistry for the derivatization and reaction of polysaccharides and monosaccharides such as those described earlier.
Carbohydrates may be purified from the natural sources or synthesized according to conventional procedures. Carbohydrates are available commercially from many suppliers.
In another aspect, the invention relates to an immunogenic vaccine against disease states particularly human disease and in particular against tumour cells expressing mucin or a subunit thereof, which vaccine comprises the immunogenic peptide, protein or portion thereof described in an earlier aspect of the invention optionally with an adjuvant in association with a pharmaceutically acceptable carrier.
The adjuvant may be any suitable adjuvant known in the art such as Quil A. QS-21 Iscoms, liposomes, alum, salts, oil, emulsions, etc.
The immunogenic peptide, protein or portion thereof may also be used to pulse dendritic cells for in vivo transfer and use as a vaccine.
In yet another aspect the invention relates to an immunogenic vaccine against disease states, particularly human disease and in particular against tumour cells expressing mucin or a subunit thereof, which vaccine comprises the immunogenic peptide, protein or portion thereof conjugated to a carbohydrate polymer as described in an earlier aspect of the invention optionally in association with a pharmaceutically acceptable carrier.
In yet another aspect of the invention, there is provided a method for inducing a cell mediated immune response against mucin which comprises administering to an animal (including a human) the immunogenic peptide, protein or portion thereof described in an earlier aspect of the invention, optionally in association with a pharmaceutically acceptable carrier.
In a still further aspect of this invention, there is provided a method for inducing a cell mediated immune response against mucin which comprises administering to an animal (including a human) a compound comprising a conjugate between said immunogenic peptide, protein or portion thereof and a carbohydrate polymer as described in an earlier aspect of the invention, optionally in association with a pharmaceutically acceptable carrier.
The immunization of humans and animals with the immunogenic peptide, protein or portion thereof or carbohydrate conjugate compounds of this invention may provoke a potentiated cellular response of activated T-lymphocytes which are cytotoxic to cells expressing the antigen component as described earlier.
The immunogenic peptide, protein, portion thereof or carbohydrate conjugate compounds of the invention may also be used as therapeutic agents for the treatment of patients suffering from cancer, as a part of the overall treatment for eradication of the cancer as described earlier.
In still a further aspect, the invention relates to the use of immunogenic peptide, protein, portion thereof or a conjugate thereof with a carbohydrate polymer in the treatment of adenocarcinoma, particularly breast cancer.
The immunogenic peptide, protein, portion thereof or carbohydrate conjugate compounds of this invention possess the advantage of being substantially non-toxic on administration to animals or humans, and as a consequence the compounds are well tolerated by administration to patients.
In yet another aspect the invention provides isolated nucleic acid sequences encoding the immunogenic peptide, protein or portion thereof described in an earlier aspect of the invention and includes vectors containing such nucleic acid sequences. The nucleic acids may be used as a basis for DNA vaccines. Such nucleic acids may be generated according to standard techniques either by cloning or synthesis as in Sambrook et al (7).
In yet another aspect the invention provides a compound comprising a conjugate between native MUC1 and a carbohydrate polymer. Preferably the native MUC1 is HMFG. This provides an advantage in that a greater number of epitopes or antigens are presented. This means that the compound may be immunogenic in a larger number of people depending on their HLA subtypes. HMGF used as a basis for the compound may be isolated and purified from natural sources which include but are not limited to body fluids such as breast milk, serum and ascites. The HMFG may also be a recombinant protein preferably produced by a eukaryotic cell.
The invention will now be described with reference to the following non-limiting Examples.
The following abbreviations are used in the Examples:
ELISA: enzyme linked immunosorbent assay
DTH: delayed type hypersensitivity
FP: fusion protein
GST: glutathione-S-transferase
HMFG: human milk fat globule
Kd: kilodalton
KLH: keyhole-limpet haemocyanin
PAGE: polyacrylamide gel electrophoresis
PBS: phosphate buffered saline
SDS: sodium dodecyl sulphate
Tc: cytotoxic T-lymphocytes
VNTR: variable number of tandem repeats
CTL: cytotoxic T-cells
M-FP: mannan fusion protein
MHC: major histocompatability complex
MSA: mucin serum antigen
CASA: circulating MUC1 serum antigen